Uridine diphosphate-dependent glycosyltransferase enzyme

ABSTRACT

In various aspects, the present invention provides uridine diphosphate-dependent glycosyltransferase (UGT) enzymes capable of catalyzing the transfer of a monosaccharide moiety from a NDP-sugar to the 3′ carbon of a sugar moiety of a substrate, such as a terpenoid glycan, thereby functioning as a “1-3 UGT.” In other aspects, the invention provides polynucleotides encoding the 1-3 UGT, and host cells comprising the same. In still other aspects, the invention provides methods for preparing glycosylated substrates, including steviol glycosides, using the enzyme and host cells of this disclosure.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/866,148, filed Jun. 25, 2019, the entire disclosureof which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to enzymes, encoding polynucleotides,host cells, and methods for producing glycosylated substrates.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: a computer readableformat copy of the Sequence Listing (filename: MAN-024PC_SequenceListing_ST25.txt; date recorded: Jun. 24, 2020; file size: 28,457bytes).

BACKGROUND

High intensity sweeteners possess a sweetness level that is many timesgreater than the sweetness level of sucrose. They are essentiallynon-caloric and are commonly used in diet and reduced-calorie products,including foods and beverages. High intensity sweeteners do not elicit aglycemic response, making them suitable for use in products targeted todiabetics and others interested in controlling their intake ofcarbohydrates.

Steviol glycosides are a class of compounds found in the leaves ofStevia rebaudiana Bertoni, a perennial shrub of the Asteraceae(Compositae) family native to certain regions of South America. They arecharacterized structurally by a single base, steviol, differing by thepresence of carbohydrate residues at positions C13 and C19. Theyaccumulate in Stevia leaves, composing approximately 10% to 20% of thetotal dry weight. On a dry weight basis, the four major glycosides foundin the leaves of Stevia typically include stevioside (9.1%),Rebaudioside A (3.8%), Rebaudioside C (0.6-1.0%) and dulcoside A (0.3%).Other known steviol glycosides include Rebaudiosides B, C, D, E, F andM, steviolbioside and rubusoside.

The minor glycosylation product Rebaudioside M (RebM) is estimated to beabout 200-350 times more potent than sucrose, and is described aspossessing a clean, sweet taste with a slightly bitter or licoriceaftertaste. Prakash I. et al., Development of Next Generation SteviaSweetener: Rebaudioside M, Foods 3(1), 162-175 (2014). Reb M is of greatinterest to the global food industry.

Processes for preparing steviol glycosides from the stevia plant are notsustainable, and are not suitable for providing the minor glycosylationproducts of stevia leaves. Accordingly, there remains a need forsustainable and economical methods for preparing compositions comprisingsteviol glycosides, including highly purified steviol glycosidecompositions. Further, methods are needed for producing substantialamounts of the minor glycosylation products, such as RebM, and others.

SUMMARY OF THE INVENTION

In various aspects, the present invention provides uridine diphosphate(UDP)-dependent glycosyltransferase (UGT) enzymes capable of catalyzingthe transfer of a monosaccharide moiety from an NDP-sugar (e.g.,UDP-sugar) to the 3′ carbon of a sugar moiety of a substrate, such as aterpenoid glycan, thereby functioning as a “1-3 UGT.” In other aspects,the invention provides polynucleotides encoding the 1-3 UGT, and hostcells comprising the same. In still other aspects, the inventionprovides methods for preparing glycosylated substrates, includingsteviol glycosides, using the enzyme and host cells of this disclosure.

The 1-3 UGT exhibits high glycosyltransferase activity on terpenoidglycosides, such as steviol glycosides. For example, the 1-3 UGTcatalyzes transfer of a monosaccharide moiety from an NDP-sugar to the3′ carbon of a sugar moiety on a terpenoid glycan, such as steviosideand RebD. That is, where the substrate is a steviol glycoside, the 1-3UGT can catalyze NDP-dependent transfer of a monosaccharide moiety tothe 3′ carbon of both the C13- or C19-linked glucose moieties. Invarious embodiments, the 1-3 UGT catalyzes the biosynthesis of RebM.

In one aspect, the invention provides a 1-3 UGT enzyme comprising anamino acid sequence that is at least about 75% identical to the aminoacid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, theenzyme comprises an amino acid substitution at positions correspondingto positions 29, 200, 357, and 414 of SEQ ID NO: 1 (Stevia rebaudianaUGT76G1). Substitutions at these positions, which are included in theenzyme of SEQ ID NOS: 5 and 6 (positions 183, 354, 54, and 111,respectfully, in SEQ ID NO: 5) can provide dramatic improvements inactivity relative to the enzyme of SEQ ID NO: 1.

In some embodiments, the 1-3 UGT enzyme comprises an insertion (withrespect to SEQ ID NO: 1) of from 5 to about 15 amino acids, or about 6to about 12 amino acids, after the position corresponding to position155 of SEQ ID NO: 5. In some embodiments, the insertion is a flexibleand hydrophilic sequence, which may be predominately Glycine and Serineresidues. In some embodiments, the sequence is GSGGSG (SEQ ID NO: 7) orGSGGSGGSG (SEQ ID NO: 8).

In various embodiments, the 1-3 UGT enzyme shows improved conversion ofstevioside to Reb A, and improved conversion of RebD to RebM, ascompared to UGT76G1-L200A (SEQ ID NO: 3).

In some embodiments, the identity of amino acids at positionscorresponding to positions 183, 354, 54, and 111 of SEQ ID NO: 5, allowsfor further modification at other positions. For example, in someembodiments, the 1-3 UGT enzyme comprises an amino acid sequence that isat least about 60% identical to the amino acid sequence of SEQ ID NO: 5,wherein the UGT enzyme comprises: a glycine (G) or threonine (T) at theposition corresponding to position 54 of SEQ ID NO: 5; a leucine (L) orisoleucine (I) at the position corresponding to position 111 of SEQ IDNO: 5; a methionine (M) or leucine (L) at the position corresponding toposition 183 of SEQ ID NO: 5; and an alanine (A), or glycine (G), orserine (S) at the position corresponding to position 354 of SEQ ID NO:5. In some embodiments, the 1-3 UGT enzyme comprises a methionine (M) atthe position corresponding to position 183 of SEQ ID NO: 5. In someembodiments, the 1-3 UGT enzyme comprises a glycine (G) at the positioncorresponding to position 54 of SEQ ID NO: 5. In some embodiments, the1-3 UGT enzyme comprises a leucine (L) at the position corresponding toposition 111 of SEQ ID NO: 5. In some embodiments, the 1-3 UGT has twoor three of a methionine (M) at the position corresponding to position183 of SEQ ID NO: 5, a glycine (G) at the position corresponding toposition 54 of SEQ ID NO: 5, and a leucine (L) at the positioncorresponding to position 111 of SEQ ID NO: 5. These modifications canprovide substantial improvements to the activity of the enzyme, relativeto the amino acid identity at the corresponding position of SEQ ID NO:1.

In some embodiments, the 1-3 UGT enzyme includes one or more of: adeletion of amino residues 159 to 161 with reference to the amino acidsequence of SEQ ID NO: 5, a substitution at position 262 (e.g., L262Q),a substitution at position 294 (e.g., R294), and a substitution atposition 413 (e.g., D413E), in each case with reference to the aminoacid sequence of SEQ ID NO: 5.

In embodiments, the 1-3 UGT enzyme includes a deletion of amino residues159 to 161 as well as the amino acid substitutions L262Q, R294P andD413E, each with reference to the amino acid sequence of SEQ ID NO: 5.An exemplary UGT enzyme according to these embodiments is disclosedherein as SEQ ID NO: 6).

In some embodiments, the 1-3 UGT enzyme includes a deletion of residuesof one or more of residues E225 to T232 with reference to the amino acidsequence of SEQ ID NO: 6. In these or other embodiments, the 1-3 UGTenzyme includes one or more amino acid substitutions at positionsselected from position 72 (e.g., S72Q), position 305 (e.g., A305C),position 345 (e.g., Y345F), and position 428 (e.g., L428I), in each casewith reference to the amino acid sequence of SEQ ID NO: 6. In exemplaryembodiments, the 1-3 UGT enzyme includes a deletion of amino residuesE225 to T232 as well as the amino acid substitutions S72Q, A305C, Y345F,and L428I, in each case with reference to the amino acid sequence of SEQID NO: 6. An exemplary UGT enzyme in accordance with these embodimentsis disclosed herein as SEQ ID NO: 9).

In some embodiments, the amino acid modifications described herein arealternatively applied to SrUGT76G1, or circular permutants thereof.

In other aspects, the invention provides polynucleotides encoding the1-3 UGT enzyme disclosed herein, as well as host cells comprising thesame. The host cell may be a microorganism, a fungal cell, an algalcell, or a plant cell. The plant may be a stevia plant or, moreparticularly, a Stevia rebaudiana plant. Stevia plants naturally expressthe enzymes required to synthesize steviol and steviol glycosides, butthey produce only trace amounts of highly glycosylated steviolglycosides such as RebM. In contrast, the RebM content of a stevia orStevia rebaudiana plant expressing a polynucleotide encoding the 1-3 UGTmay produce comparatively high levels of RebA, RebD, and/or RebM; orother typically minor steviol glycosides comprising a 1-3 glycosylationsuch as RebB, RebG, RebI, and Reb4. In various embodiments, the cell isa microbial cell, such as E. coli.

In the various embodiments, the host cell may express one or moreadditional UGT enzymes selected from C-13 UGT enzyme, a C-19 UGT enzyme,and a 1-2 UGT enzyme. “C-13 UGT” or UGTc13 is a glycosyltransferasecapable of glycosylating steviol or a steviol glycoside at its C13hydroxyl group. “C-19 UGT” or UGTc19 is a glycosyltransferase capable ofglycosylating steviol or a steviol glycoside at its C19 carboxyl group.“1-2 UGT” or UGT1-2 is a glycosyltransferase capable of β1,2glycosylation of the C2′ of a 13-O-glucose, and/or a 19-O-glucose. “1-3UGT” or UGT1-3 is a glycosyltransferase capable of β1,3 glycosylation ofthe C3′ of a 13-O-glucose, and/or a 19-O-glucose.

In some embodiments, the host cell expresses (in addition to the 1-3 UGTenzyme) a heterologous C-13 UGT enzyme, a heterologous C-19 UGT enzyme,and a heterologous 1-2 UGT enzyme, and is thus capable of glycosylatingsteviol and steviol glycoside substrates to produce RebM.

In some embodiments, the host cell (e.g., bacterial or yeast cell)produces steviol substrate by expression of endogenous and/orheterologous enzymes for biosynthesis of steviol. In these embodiments,the cell produces steviol glycosides, such as RebM, from carbon sourcessuch as glucose, sucrose, or glycerol, among others.

In some aspects, the invention provides a method for transferring amonosaccharide group to a substrate. The method comprises contacting anNDP-sugar (e.g., UDP-monosaccharide) and the substrate with the 1-3 UGTenzyme described herein, or with a host cell expressing the 1-3 UGTenzyme or a lysate thereof. Various substrates can be glycosylatedaccording to this disclosure, including but not limited to terpenoids.In some embodiments, the substrate is a terpenoid substrate, and can bea diterpenoid, such as steviol and/or steviol glycosides. In someembodiments, the substrate comprises stevioside and/or RebD. In variousembodiments, the nucleotide diphosphate is UDP or ADP, or other NDPcapable of acting as a glycosyl donor molecule. In various embodiments,the monosaccharide is glucose, galactose, fructose, rhamnose, or xylose.In some embodiments, the monosaccharide is glucose. The NDP-sugar can besupplied exogenously for in vitro reactions, or is produced endogenouslyby a host cell for embodiments that employ microbial fermentation orbiotransformation reactions. Various modifications can be made to thehost cell to increase available UDP-glucose to support the reactions.

In some embodiments, the substrate comprises a plant extract, which isoptionally a stevia leaf extract. In various embodiments, a microbialcell expressing the 1-3 UGT may be fed with steviol or a lower ordersteviol glycoside for the production of higher order steviol glycosides,including RebD and RebM. Advanced intermediates from stevia leaf extractare readily available from existing industrial extraction of steviolglycosides.

In various embodiments, the 1-3 UGT converts a lower order steviolglycoside to a higher order steviol glycoside. For example, the UGTenzyme may have 1-3′ UGT activity for the conversion of steviobioside toRebB, rubusoside to RebG, stevioside to Reb A, Reb A to RebI, RebG toReb4, RebE to Reb D, and/or Reb D to Reb M. Alternatively, the UGT maybe used in combination with another 1-3 UGT enzyme or enzymes having apreference of specificity for certain substrates. For example, one UGTmay preferentially act as a 1-3 UGT on C13 glycosyl substrates whereasanother UGT enzyme preferentially acts as a 1-3 UGT on C19 glycosylsubstrates.

In some embodiments, the method comprises growing the host cell in thepresence of the substrate. The substrate may be fed to the culture, orin some embodiments, the substrate is synthesized by the host cell. Insome embodiments, the substrate comprises steviol or comprises a mixtureof stevioside and RebA as major components, and the host cell expressesa plurality of UGT enzymes to produce target steviol glycosides, such asRebM.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structure of Rebaudioside M (RebM), a minorcomponent of the steviol glycoside family. RebM is a derivative of thediterpenoid steviol (box) with six glucosyl modifications.

FIG. 2 shows glycosylation pathways from steviol to RebM and othersteviol glycosides. UGTc13 is a glycosyltransferase capable ofglycosylating steviol or a steviol glycoside at its C13 hydroxyl group.UGTc19 is a glycosyltransferase capable of glycosylating steviol or asteviol glycoside at its C19 carboxyl group. UGT1-2 is aglycosyltransferase capable of β1,2 glycosylation of the C2′ of a13-O-glucose, and/or a 19-O-glucose. UGT1-3 is a glycosyltransferasecapable of β1,3 glycosylation of the C3′ of a 13-O-glucose, and/or a19-O-glucose.

FIG. 3A-FIG. 3B show amino acid sequence alignment of SrUGT76G1 (SEQ IDNO: 1) and MbUGT1-3_2 (SEQ ID NO: 6), which is a circularly permutedversion of SrUGT76G1. FIG. 3A shows the N-terminal portion of SrUGT76G1aligned to the C-terminal portion of MbUGT1-3_2. FIG. 3B shows theC-terminal portion of SrUGT76G1 aligned to the N-terminal portion ofMbUGT1-3_2.

FIG. 4 shows the percent conversion of stevioside to RebA, and percentconversion of RebD to RebM in vitro by the followingglycosyltransferases: UGT76G1-L200A (SEQ ID NO: 3), MbUGT1-3_0 (SEQ IDNO: 4), MbUGT1-3_1 (SEQ ID NO: 5), and MbUGT1-3_2 (SEQ ID NO: 6).

FIG. 5 shows the fold improvement for conversion of stevioside to RebAand for conversion of RebD to RebM, compared to UGT76G1-L200A.MbUGT1-3_1 and MbUGT1-3_2 show very stark improvements in enzymeproductivity for both conversions.

DETAILED DESCRIPTION OF THE INVENTION

In various aspects, the present invention provides uridinediphosphate-dependent glycosyltransferase (UGT) enzymes capable ofcatalyzing the transfer of a monosaccharide moiety from an NDP-sugar(e.g., UGT-glucose) to the 3′ carbon of a sugar moiety of a substrate,such as a terpenoid glycan, thereby functioning as a “1-3 UGT.” In otheraspects, the invention provides polynucleotides encoding the 1-3 UGT,and host cells comprising the same. In still other aspects, theinvention provides methods for preparing glycosylated substrates,including steviol glycosides, using the enzyme and host cells of thisdisclosure.

The 1-3 UGT enzyme exhibits high glycosyltransferase activity onterpenoid glycosides, such as steviol glycosides. For example, the 1-3UGT enzyme catalyzes transfer of a monosaccharide moiety from anNDP-sugar to the 3′ carbon of a sugar moiety on a terpenoid glycan, suchas stevioside and RebD. That is, where the substrate is a steviolglycoside, the 1-3 UGT can catalyze NDP-dependent transfer of amonosaccharide moiety to the 3′ carbon of both the C13- or C19-linkedsugar (e.g., glucose) moieties. In some embodiments, the 1-3 UGT enzymehas a higher rate or productivity for glycosylation at the C19-linkedsugar, as compared to at the C13-linked sugar. In some embodiments, themonosaccharide is glucose, but in other embodiments, the monosaccharidemay be galactose, fructose, rhamnose, xylose, or other monosaccharide.

In various embodiments, the 1-3 UGT catalyzes the biosynthesis of RebM.The structure of RebM is shown in FIG. 1. RebM comprises a steviolscaffold with six glycosylations: (1) a C13 O-glycosylation, (2) a C131-2 glycosylation, (3) a C13 1-3 glycosylation, (4) a C19O-glycosylation, (5) a C19 1-2 glycosylation, and (6) a C19 1-3glycosylation. As shown in FIG. 2, the 1-3 UGT can produce RebM throughthe transfer of an additional glucose to RebD substrate. Further, the1-3 UGT enzyme can catalyze the transfer of a monosaccharide to othersteviol glycoside substrates, producing products such as RebB (fromsteviolbioside), RebG (from rubusoside), Reb4 (from RebG), RebA (fromstevioside), RebD (from RebE), and RebI (from RebA). In someembodiments, the substrate comprises a plant extract, such as a stevialeaf extract, and the 1-3 UGT (along with the action of otherglycosyltransferase enzymes) is able to glycosylate the various major orminor glycosylation products to produce a product that is predominatelyRebM.

In one aspect, the invention provides a 1-3 UGT enzyme comprising anamino acid sequence that is at least about 75% identical to the aminoacid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the1-3 UGT comprises an amino acid sequence that is at least about 80%identical to SEQ ID NO: 5 or 6. In some embodiments, the amino acidsequence is at least about 85% identical to SEQ ID NO: 5 or 6, or atleast about 90% identical to SEQ ID NO: 5 or 6, or at least about 95%identical to SEQ ID NO: 5 or 6, or at least about 98% identical to SEQID NO: 5 or 6. In some embodiments, the amino acid sequence comprisesthe amino acid of SEQ ID NO: 5 or 6.

In some embodiments, the 1-3 UGT enzyme comprises an amino acid sequencethat is at least about 75% identical to the amino acid sequence of SEQID NO: 9. In some embodiments, the 1-3 UGT comprises an amino acidsequence that is at least about 80% identical to SEQ ID NO: 9. In someembodiments, the amino acid sequence is at least about 85% identical toSEQ ID NO: 9, or at least about 90% identical to SEQ ID NO: 9, or atleast about 95% identical to SEQ ID NO: 9, or at least about 98%identical to SEQ ID NO: 9. In some embodiments, the amino acid sequencecomprises the amino acid of SEQ ID NO: 9.

For example, the amino acid sequence may have from 1 to 20 amino acidmodifications independently selected from substitutions, deletions, andinsertions, with respect to the amino acid sequence SEQ ID NO: 5 or 6.In some embodiments, the amino acid sequence has from 1 to 10 amino acidmodifications (e.g., from 1 to 5) independently selected fromsubstitutions, deletions, and insertions, with respect to the amino acidsequence of SEQ ID NO: 5 or 6. In some embodiments, the UGT amino acidsequence has from 1 to 20 amino acid modifications independentlyselected from substitutions, deletions, and insertions, with respect tothe amino acid sequence of SEQ ID NO: 9. In some embodiments, the aminoacid sequence has from 1 to 10 amino acid modifications (e.g., from 1 to5) independently selected from substitutions, deletions, and insertions,with respect to the amino acid sequence of SEQ ID NO: 9. Amino acidmodifications to the amino acid sequence of SEQ ID NO: 5, 6, or 9, canbe guided by available enzyme structures and construction of homologymodels. Exemplary structures are described in, e.g., Li, et al.,“Crystal Structure of Medicago truncatula UGT85H2—insights into theStructural Basis of a Multifunctional (iso) FlavonoidGlycosyltransferase,” J. of Mol. Biol. 370.5 (2007): 951-963 as well asLee et al., “Molecular Basis for Branched Steviol GlucosideBiosynthesis,” PNAS 116:13131-13136 (2019). Publicly available crystalstructures (e.g., PDB entry: 2PQ6) may be used to inform amino acidmodifications. For example, one or more amino acid modifications can bemade to the active site or in the vicinity of the active site to improvethe binding of substrate, and/or to improve reaction geometries of thesesubstrates with catalytic side chains.

In some embodiments, the enzyme comprises an amino acid substitution atpositions corresponding to positions 29, 200, 357, and 414 of SEQ ID NO:1 (Stevia rebaudiana UGT76G1). Substitutions at these positions, whichare included in the enzyme of SEQ ID NOS: 5, 6, and 9 (positions 183,354, 54, and 111, respectfully, in SEQ ID NO: 5) can provide dramaticimprovements in activity. In some embodiments, the identity of aminoacids at positions corresponding to positions 183, 354, 54, and 111 ofSEQ ID NO: 5, allows for further modification at other positions. Forexample, in some embodiments, the 1-3 UGT enzyme comprises an amino acidsequence that is at least about 60% identical to the amino acid sequenceof SEQ ID NO: 5, 6, or 9, wherein the UGT enzyme comprises: a glycine(G) or threonine (T) at the position corresponding to position 54 of SEQID NO: 5; a leucine (L) or isoleucine (I) at the position correspondingto position 111 of SEQ ID NO: 5; a methionine (M) or leucine (L) at theposition corresponding to position 183 of SEQ ID NO: 5; and an alanine(A), or glycine (G), or serine (S) at the position corresponding toposition 354 of SEQ ID NO: 5. In some embodiments, the 1-3 UGT enzymecomprises a methionine (M) at the position corresponding to position 183of SEQ ID NO: 5. In some embodiments, the 1-3 UGT enzyme comprises aglycine (G) at the position corresponding to position 54 of SEQ ID NO:5. In some embodiments, the 1-3 UGT enzyme comprises a leucine (L) atthe position corresponding to position 111 of SEQ ID NO: 5. In someembodiments, the 1-3 UGT has two or three of a methionine (M) at theposition corresponding to position 183 of SEQ ID NO: 5, a glycine (G) atthe position corresponding to position 54 of SEQ ID NO: 5, and a leucine(L) at the position corresponding to position 111 of SEQ ID NO: 5. Thesemodifications can provide substantial improvements to the activity ofthe enzyme.

The 1-3 UGT enzyme may comprise other substitutions at the positioncorresponding to position 54 of SEQ ID NO: 5, other than Serine, whichis at the corresponding position (357) of SEQ ID NO: 1. Thus, in someembodiments, the substitution at the position corresponding to position54 of SEQ ID NO: 5 is a hydrophobic amino acid, such as alanine (A),valine (V), leucine (L), isoleucine (I), phenylalanine (F), ormethionine (M). In some embodiments, the amino acid at the positioncorresponding to position 54 of SEQ ID NO: 5 has a side chain that doesnot have the ability to provide a hydrogen bond. In other embodiments,the amino acid at the position corresponding to position 54 of SEQ IDNO: 5 is Glycine (G), asparagine (N), cysteine (C), glutamine (Q),threonine (T), or tyrosine (Y).

In various embodiments, the 1-3 UGT enzyme comprises a leucine (L) aminoacid at the position corresponding to position 111 of SEQ ID NO: 5. Insome embodiments, the 1-3 UGT enzyme may comprise an amino acid otherthan leucine at this position. In various embodiments, the positioncorresponding to position 111 of SEQ ID NO: 5 will not be Valine, theamino acid at the corresponding position of SEQ ID NO: 1. Other suitablesubstitutions at the position corresponding to position 111 may includeglycine (G), alanine (A), isoleucine (I) or methionine (M). In someembodiments, the amino acid at the position corresponding to position111 of SEQ ID NO: 5 has a side chain that is less hydrophobic and/orbulky than valine.

In various embodiments, the UGT comprises a methionine (M) amino acid atthe position corresponding to position 183 of SEQ ID NO: 5. In variousembodiments, the UGT comprises another suitable amino acid at thisposition, other than isoleucine, which is at the corresponding positionof SEQ ID NO: 1. For example, the amino acid at this position may have aside chain that is less hydrophobic than isoleucine and/or may provide ahydrogen bond. Some exemplary substitutions at the positioncorresponding to position 183 of SEQ ID NO: 5 include alanine (A),valine (V), leucine (L), Cysteine (C), Serine (S), Threonine (T),Tyrosine (Y), Asparagine (N), Glutamine (Q), Aspartic Acid (D), andGlutamic acid (E).

In various embodiments, the 1-3 UGT comprises an Alanine (A) or Glycine(G) at the position corresponding to position 354 of SEQ ID NO: 5. Invarious embodiments, the amino acid at this position has a side chainthat is less hydrophobic and/or less bulky than leucine, which is at thecorresponding position of SEQ ID NO: 1.

In some embodiments, the 1-3 UGT enzyme comprises an insertion (withrespect to UGT76G1, SEQ ID NO: 1) of from 5 to about 15 amino acids,such as from 6 to 12 amino acids, or about 6 or about 11 amino acids,after the position corresponding to position 155 of SEQ ID NO: 5. Insome embodiments, the insertion is a flexible and hydrophilic sequence,that is predominately Glycine and Serine residues. In some embodiments,the sequence is GSGGSG (SEQ ID NO: 7) or GSGGSGGSG (SEQ ID NO: 8).

In various embodiments, the 1-3 UGT enzyme shows improved conversion ofstevioside to Reb A, and improved conversion of RebD to RebM, ascompared to UGT76G1-L200A (SEQ ID NO: 3). This improved conversion isexhibited in a bioconversion assay where stevioside or RebD substrate isfed to microbial cells expressing the 1-3 UGT enzyme of this disclosure.Improved conversion can be demonstrated in reactions with cell lysatescontaining recombinantly expressed 1-3 UGT, or in vitro reactions withpurified or partially purified 1-3 UGT. Such reactions are well known inthe art. Alternatively, whole cell assays can be used. For example, anE. coli strain (ΔushA, ΔgalETKM, Δpgi; overexpressed pgm, galU)expressing the 1-3 UGT enzyme is grown overnight in 96-well plates at250 rpm at 37° C. The cells are then transferred to a fresh productionculture to 10% of the total volume. 0.5 mM substrate (e.g., steviosideor Rebaudioside D) are included in the production culture. Theproduction culture is then grown for 48 hours in 96-well plates at 250rpm at 37° C. Products can be quantified using a LC-MS QQQ.

In some embodiments, the identity of amino acids at positionscorresponding to positions 183, 354, 54, and 111 of SEQ ID NO: 5, allowsfor further modification at other positions. For example, in someembodiments, the 1-3 UGT enzyme comprises an amino acid sequence that isat least about 60% identical to the amino acid sequence of SEQ ID NO: 5,6, or 9, wherein the UGT enzyme comprises: a glycine (G) or threonine(T) at the position corresponding to position 54 of SEQ ID NO: 5; aleucine (L) or isoleucine (I) at the position corresponding to position111 of SEQ ID NO: 5; a methionine (M) or leucine (L) at the positioncorresponding to position 183 of SEQ ID NO: 5; and an alanine (A), orglycine (G), or serine (S) at the position corresponding to position 354of SEQ ID NO: 5. In some embodiments, the 1-3 UGT enzyme comprises amethionine (M) at the position corresponding to position 183 of SEQ IDNO: 5. In some embodiments, the 1-3 UGT enzyme comprises a glycine (G)at the position corresponding to position 54 of SEQ ID NO: 5. In someembodiments, the 1-3 UGT enzyme comprises a leucine (L) at the positioncorresponding to position 111 of SEQ ID NO: 5. In some embodiments, the1-3 UGT has two or three of a methionine (M) at the positioncorresponding to position 183 of SEQ ID NO: 5, a glycine (G) at theposition corresponding to position 54 of SEQ ID NO: 5, and a leucine (L)at the position corresponding to position 111 of SEQ ID NO: 5. Thesemodifications can provide substantial improvements to the activity ofthe enzyme. In some embodiments, the amino acid sequence is at leastabout 70% identical to the amino acid sequence of SEQ ID NO: 5, 6, or 9,or at least about 80% identical to the amino acid sequence of SEQ ID NO:5, 6, or 9, or at least about 90% identical to the amino acid sequenceof SEQ ID NO: 5, 6, or 9, or at least about 95% to the amino acidsequence of SEQ ID NO: 5, 6, or 9.

In some embodiments, the 1-3 UGT enzyme comprises a glycine (G) aminoacid at the position corresponding to position 54 of SEQ ID NO: 5, aleucine (L) amino acid at the position corresponding to position 111 ofSEQ ID NO: 5; and a methionine (M) amino acid at the positioncorresponding to position 183 of SEQ ID NO: 5.

In some embodiments, the 1-3 UGT enzyme comprises a glycine (G) aminoacid at the position corresponding to position 54 of SEQ ID NO: 5, and aleucine (L) amino acid at the position corresponding to position 111 ofSEQ ID NO: 5.

In some embodiments, the 1-3 UGT enzyme comprises a glycine (G) aminoacid at the position corresponding to position 54 of SEQ ID NO: 5, and amethionine (M) amino acid at the position corresponding to position 183of SEQ ID NO: 5.

In some embodiments, the 1-3 UGT enzyme comprises a leucine (L) aminoacid at the position corresponding to position 111 of SEQ ID NO: 5 and amethionine (M) amino acid at the position corresponding to position 183of SEQ ID NO: 5.

In these or other embodiments, the 1-3 UGT enzyme has a deletion of oneor more amino acids at positions E225 to T232, with respect to the aminoacid sequence of SEQ ID NO: 6. For example, the UGT may have a deletionof at least two, at least three, at least four, at least five, at leastsix, at least seven, or eight amino acids corresponding to amino acidsE225-T232 of SEQ ID NO: 6.

In these or other embodiments, the 1-3 UGT enzyme further includes oneor more of amino acid substitutions at positions corresponding toposition 72, position 305, position 345, and position 428. For example,1-3 UGT enzyme may have a substitution of glutamine (Q) or asparagine(N) at the position corresponding to position 72 of SEQ ID NO: 6. Insome embodiments, the 1-3 UGT enzyme has a substitution of a neutralhydrophilic amino acid, such as cysteine (C), Serine (S), or Threonine(T) at the position corresponding to position 305 of SEQ ID NO: 6. Insome embodiments, the 1-3 UGT enzyme has a substitution of phenylalanine(F) or Tryptophan (W) at the position corresponding to position 345 ofSEQ ID NO: 6. In some embodiments, the 1-3 UGT enzyme has a substitutionof isoleucine (I), valine (V), or alanine (A) at the positioncorresponding to position 428 of SEQ ID NO: 6. In some embodiments, the1-3 UGT enzyme has 2, 3, or 4 of the following substitutions withrespect to SEQ ID NO: 6: S72Q, A305C, Y345F, and L428I.

In some embodiments, the 1-3 UGT enzyme comprises an insertion (withrespect to 76G1) of from 5 to about 15 amino acids, such as from about 6to about 12 amino acids, or about 6 or about 11 amino acids, after theposition corresponding to positions 155 of SEQ ID NO: 5. In someembodiments, the insertion is a flexible and hydrophilic sequence, suchas an amino acid sequence that is predominately Glycine and Serineresidues. In some embodiments, the sequence is GSGGSG (SEQ ID NO: 7) orGSGGSGGSG (SEQ ID NO: 8).

In still other aspects and embodiments, the 1-3 UGT enzyme is not acircular permutant of UGT76G1, that is, the enzyme comprises an aminoacid sequence that has at least about 75% sequence identity to the aminoacid sequence of SEQ ID NO:1. In such embodiments, the enzyme mayinclude one or more amino acid modifications described herein. Exemplarymodifications of UGT76G1 (SEQ ID NO:1) include modifications withrespect to SEQ ID NO:1 selected from: (i) a deletion of one or moreamino acids corresponding to E74 to T81, (ii) a substitution at theposition corresponding to position 357, which is optionally glycine,(iii) a substitution at the position corresponding to position 414, andwhich is optionally leucine, (iv) a substitution at the positioncorresponding to position 29, and which is optionally methionine, (v) asubstitution at the position corresponding to position 402, which isoptionally glutamine, (vi) a substitution at the position correspondingto position 154, which is optionally cysteine, (vii) a substitution atthe position corresponding to position 194, and which is optionallyphenylalanine, (viii) a substitution at the position corresponding toposition 277, and which is optionally isoleucine, (ix) a substitution atthe position corresponding to position 208, and which is optionallyglutamine, (x) a substitution at the position corresponding to position140, and which is optionally proline, and (xi) a substitution at theposition corresponding to position 259, and which is optionally glutamicacid.

For example, in some embodiments, the UGT enzyme comprises a deletion ofat least two, or at least three, at least four, at least five, at leastsix, at least seven, or all eight amino acids corresponding to E74 toT81 of SEQ ID NO:1. In various embodiments, the enzyme further comprisesa substitution of alanine at the position corresponding to position 200of SEQ ID NO: 1. In some embodiments, the enzyme has at least two,three, or four of: 357G, 414L, 29M, 402Q, 154C, 194F, 2771, 208Q, 140P,and 259E, each numbered according to SEQ ID NO:1.

In such embodiments, the enzyme may have is at least about 80% sequenceidentity, or at least about 85% sequence identity, or at least about 90%sequence identity, or at least about 95% sequence identity, or at leastabout 98% sequence identity to the amino acid sequence of SEQ ID NO:1.For example, the enzyme may have from 1 to 20, or from 1 to 10 aminoacid modifications independently selected from amino acid substitutions,deletions, and insertions with respect to SEQ ID NO:1.

In still other aspects and embodiments, the 1-3 UGT enzyme is a circularpermutant of SrUGT76G1 (see US 2017/0332673, which is herebyincorporated by reference in its entirety) and optionally having from 1to 20 or from 1 to 15, or from 1 to 10 amino acid modificationsindependently selected from amino acid substitutions, deletions, andinsertions with respect to the corresponding position of SEQ ID NO:1.Exemplary modifications with respect to positions of SEQ ID NO:1 can beselected from: (i) a deletion of one or more amino acids correspondingto E74 to T81 (as described herein), (ii) a substitution at the positioncorresponding to position 357, which is optionally glycine, (iii) asubstitution at the position corresponding to position 414, and which isoptionally leucine, (iv) a substitution at the position corresponding toposition 29, and which is optionally methionine, (v) a substitution atthe position corresponding to position 402, which is optionallyglutamine, (vi) a substitution at the position corresponding to position154, which is optionally cysteine, (vii) a substitution at the positioncorresponding to position 194, and which is optionally phenylalanine,(viii) a substitution at the position corresponding to position 277, andwhich is optionally isoleucine, (ix) a substitution at the positioncorresponding to position 208, and which is optionally glutamine, (x) asubstitution at the position corresponding to position 140, and which isoptionally proline, and (xi) a substitution at the positioncorresponding to position 259, and which is optionally glutamic acid. Insome embodiments, the UGT enzyme comprises a deletion of the amino acidscorresponding to E74 to T81.

Changes to the amino acid sequence of an enzyme can alter its activityor have no measurable effect. Silent changes with no measurable effectare most likely to be conservative substitutions and small insertions ordeletions on solvent-exposed surfaces that are located away from activesites and substrate-binding sites. In contrast, enzymatic activity ismore likely to be affected by non-conservative substitutions, largeinsertions or deletions, and changes within active sites,substrate-binding sites, and at buried positions important for proteinfolding or conformation. Changes that alter enzymatic activity mayincrease or decrease the reaction rate or increase or decrease theaffinity or specificity for a particular substrate. For example, changesthat increase the size of a substrate-binding site may permit an enzymeto act on larger substrates and changes that position a catalytic aminoacid side chain closer to a target site on a substrate may increase theenzymatic rate.

Knowledge of the three-dimensional structure of an enzyme and thelocation of relevant active sites, substrate-binding sites, and otherinteraction sites can facilitate the rational design of mutations andprovide mechanistic insight into the phenotype of specific changes.Plant UGTs share a highly conserved secondary and tertiary structurewhile having relatively low amino acid sequence identity. Osmani et al,Substrate specificity of plant UDP-dependent glycosyltransferasespredicted from crystal structures and homology modeling, Phytochemistry70 (2009) 325-347. The sugar acceptor and sugar donor substrates of UGTsare accommodated in a cleft formed between the N- and C-terminaldomains. Several regions of the primary sequence contribute to theformation of the substrate binding pocket including structurallyconserved domains as well as loop regions differing both with respect totheir amino acid sequence and sequence length.

Construction of UGT derivatives can be guided based on structureanalysis and homology modeling. See, Soon Goo Lee, et al., MolecularBasis for Branched Steviol Glucoside Biosynthesis, PNAS, Jun. 19, 2019.

For example, based on independent crystal structure preparation andanalysis of SrUGT76G1_L200A and an amino acid sequence alignment ofSrUGT76G1 to MbUGT3-1_1, it is predicted that the steviol core ofstevioside is close (within 4 Å) to the following residues ofMbUGT3-1_1: I244, L280, W351, A354, I357, M362, and T438. Further, theC19 1-2 glycosylation is predicted to be close (within 4 Å) to T438. Thesteviol core of RebD is predicted to be close (within 4 Å) of thefollowing hydrophobic side chains of MbUGT3-1_1: L239, M242, I244, L280,I353, A354, and I357. The C13 1-2′ glycosylation is predicted to beclose (within 4 Å) of the following hydrogen bonding side chains ofMbUGT3-1_1: S301 and D77. Positioning and amino acid content of theV341-Q352 and K355-A367 helices of MbUGT3-1_1 may be important forcatalysis as the mutation corresponding to L200A is in a loop betweenthese helices. Positions L76 and/or D77 of MbUGT3-1_1 may interact withthe C13 glycosylation of stevioside.

Substitutions of amino acids may be conservative substitutions ornon-conservative substitutions. Conservative substitutions are definedwhere the old and new amino acids have similar characteristics such assize and charge. Naturally occurring residues are divided into groupsbased on common side chain properties:

-   -   (group 1) hydrophobic (aliphatic): methionine (Met), Alanine        (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile);    -   (group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser),        Threonine (Thr), Asparagine (Asn), Glutamine (Gln);    -   (group 3) acidic: Aspartic acid (Asp), Glutamic acid (Glu);    -   (group 4) basic: Histidine (His), Lysine (Lys), Arginine (Arg);    -   (group 5) residues that influence chain orientation: Glycine        (Gly), Proline (Pro); and    -   (group 6) aromatic: Tryptophan (Trp), Tyrosine (Tyr),        Phenylalanine (Phe).

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another an amino acid of another class.

The amino acid sequence of the UGT enzyme can optionally include analanine inserted or substituted at position 2 to decrease turnover inthe cell. In various embodiments, the 1-3 UGT enzyme comprises analanine amino acid residue inserted or substituted at position 2 withrespect to SEQ ID NO: 5, 6, or 9, to provide additional stability invivo.

Identity of amino acid sequences, i.e. the percentage of sequenceidentity, can be determined via sequence alignments. Such alignments canbe carried out with several known algorithms, such as that described byKarlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA90: 5873-5877), with hmmalign (HMMER package) or with the CLUSTALalgorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994)Nucleic Acids Res. 22, 4673-80). The grade of sequence identity(sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ(or BlastX). A similar algorithm is incorporated into the BLASTN andBLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410.BLAST protein alignments may be performed with the BLASTP program,score=50, word length=3. To obtain gapped alignments for comparativepurposes, Gapped BLAST is utilized as described in Altschul et al (1997)Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs are used.

In other aspects, the invention provides polynucleotides encoding the1-3 UGT enzyme disclosed herein, as well as host cells comprising thesame. The host cell may be a microorganism, a fungal cell, an algalcell, or a plant cell. The plant may be a stevia plant or, moreparticularly, a Stevia rebaudiana plant. Stevia plants naturally expressthe enzymes required to synthesize steviol and steviol glycosides, butthey produce only trace amounts of highly glycosylated steviolglycosides such as RebM. In contrast, the RebM content of a stevia orStevia rebaudiana plant expressing a polynucleotide encoding the 1-3 UGTmay be more than about 1%, or more than about 2%, or more than about 5%,or more than about 10% of the steviol glycoside content of the plantleaves. Furthermore, the RebA content of a stevia or Stevia rebaudianaplant expressing a polynucleotide encoding the 1-3 UGT may be more thanabout 5%, or more than about 10%, or more than about 15% of the steviolglycoside content of the plant leaves.

The microbial host cell in various embodiments may be prokaryotic oreukaryotic. In some embodiments, the microbial host cell is a bacteriaselected from Escherichia spp., Bacillus spp., Corynebacterium spp.,Rhodobacter spp., Zymomonas spp., Vibrio spp., and Pseudomonas spp. Forexample, in some embodiments, the bacterial host cell is a speciesselected from Escherichia coli, Bacillus subtilis, Corynebacteriumglutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonasmobilis, Vibrio natriegens, or Pseudomonas putida. In some embodiments,the bacterial host cell is E. coli. Alternatively, the microbial cellmay be a yeast cell, such as but not limited to a species ofSaccharomyces, Pichia, or Yarrowia, including Saccharomyces cerevisiae,Pichia pastoris, and Yarrowia lipolytica.

The polynucleotides encoding the 1-3 UGT enzymes may be integrated intothe chromosome of the microbial cell, or alternatively, are expressedextrachromosomally. For example, the 1-3 UGT enzyme may be expressedfrom a bacterial artificial chromosome (BAC) or plasmid.

Expression of UGT enzymes can be tuned for optimal activity, using, forexample, gene modules (e.g., operons) or independent expression of theUGT enzymes. For example, expression of the genes or operons can beregulated through selection of promoters, such as inducible orconstitutive promoters, with different strengths (e.g., strong,intermediate, or weak). Several non-limiting examples of promoters ofdifferent strengths include Trc, T5 and T7. Additionally, expression ofgenes or operons can be regulated through manipulation of the copynumber of the gene or operon in the cell. In some embodiments, the cellexpresses a single copy of each UGT enzyme. In some embodiments,expression of genes or operons can be regulated through manipulating theorder of the genes within a module, where the genes transcribed firstare generally expressed at a higher level. In some embodiments,expression of genes or operons is regulated through integration of oneor more genes or operons into the chromosome.

Optimization of UGT expression can also be achieved through selection ofappropriate promoters and ribosomal binding sites. In some embodiments,this may include the selection of high-copy number plasmids, or single-,low- or medium-copy number plasmids. The step of transcriptiontermination can also be targeted for regulation of gene expression,through the introduction or elimination of structures such asstem-loops.

In some embodiments, the cell is a plant cell. For example, thepolynucleotide can be heterologously expressed in a stevia plant underthe control of a suitable promoter, such as a constitutive or induciblepromoter.

In the various embodiments, the host cell may express one or moreadditional UGT enzymes selected from C-13 UGT enzyme, a C-19 UGT enzyme,and a 1-2 UGT enzyme. In some embodiments, the host cell expresses (inaddition to the 1-3 UGT enzyme) a heterologous C-13 UGT enzyme, aheterologous C-19 UGT enzyme, and a heterologous 1-2 UGT enzyme, and isthus capable of glycosylating steviol and steviol glycoside substratesto produce RebM.

FIG. 2 illustrates the structures of steviol and various steviolglycosides, and identifies enzymatic activities in biosynthetic pathwaysleading from steviol to RebM. Likewise, Table 1 identifies substratesand products on biosynthetic pathways leading from steviol to RebM. Foreach substrate and product pair, Table 1 describes the type ofglycosylation and identifies enzymes with the requisite activity. Fourtypes glycosylation activity are required for production of RebM:primary glycosylation of the C13 and C19 carbons, and secondaryglycosylation at the 2′ or 3′ positions of the primary glycan. Theglycans are added one 6-carbon monosaccharide unit at a time. Thus, aprimary glycan must be conjugated to the C13 or C19 of steviol or asteviol glycoside before a secondary glycan can be conjugated to thatprimary glycan. However, the order of glycosylation of steviolglycosides is not otherwise restricted. Table 2 identifies enzymes fromvarious sources with the activities required on biosynthetic pathwaysleading from steviol to Reb M and provides references for theirnucleotide and amino acid sequences. See U.S. Patent ApplicationPublication 20170332673, which is hereby incorporated by reference inits entirety.

TABLE 1 Enzymes catalyzing reactions leading to Reb M. Substrate ProductGlycos. Enzyme 1 Enzyme 2 Enzyme 3 Enzyme 4 Steviol Steviolmonoside C13SrUGT85C2 Steviol C19-Glu-Steviol C19 SrUGT74G1 MbUGTc19 SteviolmonosideSteviolbioside 1-2′ SrUGT91D1 SrUGT91D2 OsUGT1-2 MbUGT1-2Steviolmonoside Rubusoside C19 SrUGT74G1 MbUGTc19 C19-Glu-SteviolRubusoside C13 SrUGT85C2 Steviolbioside Stevioside C19 SrUGT74G1MbUGTc19 Steviolbioside RebB 1-3′ SrUGT76G1 MbUGT1-3_1 Stevioside RebE1-2′ SrUGT91D1 SrUGT61D2 OsUGT1-2 MbUGT1-2 Stevioside Reb A 1-3′SrUGT76G1 MbUGT1-3_1 RebB Reb A C19 SrUGT74G1 MbUGTc19 RebE Reb D 1-3′SrUGT76G1 MbUGT1-3_1 Reb A Reb D 1-2′ SrUGT91D1 SrUGT91D2 OsUGT1-2MbUGT1-2 Reb D Reb M 1-3′ SrUGT76G1 MbUGT1-3_1 MbUGT1-3_2

TABLE 2 Enzyme/gene sequences for biosynthesis of steviol glycosidesGlycosylation type Enzyme Gene ID Protein ID Description C13 SrUGT85C2AY345978.1 AAR06916.1 C19 SrUGT74G1 AY345982.1 AAR06920.1 MbUGTc19 — —US 2017/0332673 1-2’ SrUGT91D1 AY345980.1 AAR06918.1 SrUGT91D2ACE87855.1 ACE87855.1 SrUGT91D2e — — US 2011/038967 OsUGT1-2NM_001057542.1 NP_001051007.2 WO 2013/022989 MbUGT1-2 — — US2017/0332673 1-3’ SrUGT76G1 FB917645.1 CAX02464.1 MbUGT1-3_1 Thisdisclosure MbUGT1-3_2 This disclosure

In some embodiments, the host cell produces steviol by expression ofendogenous and/or heterologous enzymes for biosynthesis of steviol. Thehost cell may be engineered to co-express the 1-3 UGT with other UGTenzymes active on terpenoids and terpenoid glycosides, such as a C-13UGT enzyme, a C-19 UGT enzyme, and a 1-2 UGT enzyme (see Tables 1-2).The microbial cell may additionally be engineered to co-express enzymesfor steviol biosynthesis, such as enzymes of the MEP or MVA pathways,copalyl synthase and kaurene synthase (which can be present as abifunctional enzyme in some embodiments), P450 enzymes such as kaureneoxidase and kaurenoic acid hydroxylase and P450 reductase enzymes. Table3. Pathways and enzymes for biosynthesis of steviol are disclosed in US20170332673, which is hereby incorporated by reference in its entirety.

TABLE 3 Summary of enzyme/gene sequences enabling biosynthesis ofsteviol. No. Enzyme Species Gene ID Protein ID 1 TcGGPPS Taxuscanadensis AF081514.1 AAD16018.1 2 AgGGPPS Abies grandis AF425235.2AAL17614.2 3 AnGGPPS Aspergillus nidulans XM_654104.1 XP_659196.1 4SmGGPPS Streptomyces melanosporofaciens AB448947.1 BAI44337.1 5 MbGGPPSMarine bacterium 443 n/a AAR37858.1 6 PhGGPPS Paracoccus haeundaensisn/a AAY28422.1 7 CtGGPPS Chlorobium tepidum TLS NC_002932.3 NP_661160.18 SsGGPPS Synechococcus sp. JA-3-3Ab n/a ABC98596.1 9 Ss2GGPPSSynechocystis sp. PCC 6803 n/a BAA16690.1 10 TmGGPPS Thermotoga maritimaHB8 n/a NP_227976.1 11 CgGGPPS Corynebacterium glutamicum n/aNP_601376.2 12 TtGGPPS Thermus thermophillus HB27 n/a YP_143279.1 13PcGGPPS Pyrobaculum calidifontis JCM 11548 n/a WP_011848845.1 14 SrCPPSStevia rebaudiana AF034545.1 AAB87091.1 15 EtCPPS Erwina tracheiphilan/a WP_020322919.1 16 SfCPPS Sinorhizobium fredii n/a WP_010875301.1 17SrKS Stevia rebaudiana AF097311.1 AAD34295.1 18 EtKS Erwina tracheiphilan/a WP_020322918.1 19 SfKS Sinorhizobium fredii n/a WP_010875302.1 20GfCPPS/KS Gibberella fujikuroi AB013295.1 Q9UVY5.1 21 PpCPPS/KSPhyscomitrella patens AB302933.1 BAF61135.1 22 PsCPPS/KS Phaeosphaeriasp. L487 AB003395.1 O13284.1 23 AtKO Arabidopsis thaliana NM_122491.2NP_197962.1 24 SrKO Stevia rebaudiana AY364317.1 AAQ63464.1 25 PpKOPhyscomitrella patens AB618673.1 BAK19917.1 26 AtCPR Arabidopsisthaliana X66016.1 CAA46814.1 27 SrCPR Stevia rebaudiana DQ269454.4ABB88839.2 28 AtKAH Arabidopsis thaliana NM_122399.2 NP_197872.1 29SrKAH1 Stevia rebaudiana DQ398871.3 ABD60225.1 30 SrKAH2 Steviarebaudiana n/a n/a

In some aspects, the invention provides a method for transferring amonosaccharide group to a substrate. The method comprises contacting anNDP-sugar (e.g., UDP-monosaccharide) and the substrate with the 1-3 UGTenzyme described herein, or a host cell expressing the 1-3 UGT enzyme ora lysate thereof. Various substrates can be glycosylated according tothis disclosure, including but not limited to terpenoids. In someembodiments, the substrate is a terpenoid substrate, and can be aditerpenoid, such as steviol and/or steviol glycosides. In someembodiments, the substrate comprises stevioside and/or RebD. In variousembodiments, the monosaccharide is glucose, galactose, fructose,rhamnose, or xylose. In some embodiments, the monosaccharide is glucose.In some embodiments, the method takes place in vitro with recombinant1-3 UGT, and includes exogenously added NDP-sugar (e.g., UDP-glucose orADP-glucose) reagent. In other embodiments, the 1-3 UGT is expressedrecombinantly in a cell, and the NDP-glucose is available endogenously.In these embodiments, substrate can be fed to the cells, and isavailable in the cell for the glycosyltransferase reactions.

Whole cell conversion (i.e., a “biotransformation reaction”) requiresthat substrate (e.g., glycoside intermediates) and product aretransported into and out of the cell, respectively, and that the cellprovides UDP-glucose cofactor regeneration. This is in contrast toprocesses that use enzymes from cell lysis or secretion outside thecell, which requires an exogenous NDP-glucose supply or NDP-glucoseprecursor or NDP-glucose regeneration mechanism or NDP-glucoseregeneration enzyme system. In embodiments of the present invention,catalysis (glycosylation) is carried out within live microbial cells.UDP-glucose cofactor recycling takes place using the native cellularmetabolism without requiring externally provided enzymes or the feedingof expensive substrates. In accordance with some embodiments, glycosideintermediates are transported into the cell, and product is transportedout of the cell.

US 2017/0332673 describes E. coli strains that overexpress MEP pathwayenzymes, along with a downstream steviol biosynthesis pathway, and UGTenzymes to drive production of RebM from glucose. However, these strainsdo not perform biocatalysis of fed steviol glycoside intermediates toRebM, which may be, in part, due to the inability of the host cell toimport the steviol glycoside substrate. In some embodiments, geneticmodifications to the microbial cell allow for glycosylated intermediatesto be translocated into the cell, while advanced glycosylated productsare secreted into the medium.

In some embodiments, the microbial cell has one or more geneticmodifications that increase UDP-glucose availability. In someembodiments, without wishing to be bound by theory, these modificationsmay also stress the cell for glucose availability, leading to theincreased expression of endogenous transporters to import steviolglycosides into the cell. Wild-type UDP-glucose levels in exponentiallygrowing E. coli is about 2.5 mM (Bennett B D, et al., Absolutemetabolite concentrations and implied enzyme active site occupancy inEscherichia coli. Nat Chem Biol. 2009; 5(8):593-9). In some embodiments,genetic modifications to the host cell are engineered to increaseUDP-glucose, e.g., to at least 5 mM, or at least 10 mM, in exponentiallygrowing cells (e.g., that do not have recombinant expression of UGTenzymes).

In some embodiments, the microbial cell has a deletion, inactivation, orreduced activity or expression of a gene encoding an enzyme thatconsumes UDP-glucose. For example, the microbial cell may have adeletion, inactivation, or reduced activity of ushA (UDP-sugarhydrolase) and/or one or more of galE, galT, galK, and galM (which areresponsible for UDP-galactose biosynthesis from UDP-glucose), orortholog thereof in the microbial species. In some embodiments, galETKMgenes are inactivated, deleted, or substantially reduced in expression.

In these or other embodiments, the microbial cell has a deletion,inactivation, or reduced activity or expression of a gene encoding anenzyme that consumes a precursor to UDP-glucose. For example, in someembodiments, the microbial cell has a deletion, inactivation, or reducedactivity or expression of pgi (glucose-6 phosphate isomerase), orortholog thereof in the microbial species of the host cell.

In these or other embodiments, the cell has an overexpression orincreased activity of one or more genes encoding an enzyme involved inconverting glucose-6-phosphate to UDP-glucose. For example, pgm(phosphoglucomutase) and/or galU (UTP-glucose-1-phosphateuridylyltransferase) (or ortholog or derivative thereof) can beoverexpressed, or modified to increase enzyme productivity. See, US2020/0087692, which is hereby incorporated by reference in its entirety.

In some embodiments, the substrate is a plant extract, which isoptionally a stevia leaf extract. In various embodiments, a microbialcell expressing the 1-3 UGT may be fed with steviol or a lower ordersteviol glycoside for the production of higher order steviol glycosides,including Reb D and Reb M. Advanced intermediates from stevia leafextract are readily available from existing industrial extraction ofsteviol glycosides. As shown in Table 4, available leaf extract containsprimarily the pathway intermediates stevioside and Rebaudioside A(RebA). In various embodiments, the stevia leaf extract is an extractionof steviol glycosides. In some embodiments, the extract comprises one ormore of stevioside, steviolbioside, and Rebaudioside A, as prominentcomponents. A prominent component generally makes up at least about 10%of the steviol glycosides in the extract, but in some embodiments, maymake up at least about 20%, or at least about 25%, or at least about 30%of the steviol glycosides in the extract.

TABLE 4 Steviol Glycoside Composition of Available Stevia Leaf Extract %Batch 1 Batch 2 Batch 3 Rebaudioside A 38.2 10.5 30.3 Stevioside 8.5 9.018.4 Rebaudioside C 12.9 4.2 16.6 Rebaudioside B 4.3 7.1 1.2 Rubusoside5.0 2.2 2.0 Rebaudioside F 2.0 2.7 2.1 Steviolbioside 0.3 3.7 0.3Rebaudioside D 0.2 2.1 0.9 Dulcoside A 0.9 0.4 0.5

In various embodiments, the 1-3 UGT converts a lower order steviolglycoside to a higher order steviol glycoside. For example, the UGTenzyme may have 1-3′ UGT activity for the conversion of steviobioside toRebB, rubusoside to RebG, stevioside to Reb A, Reb A to RebI, RebG toReb4, RebE to Reb D, and/or Reb D to Reb M. Alternatively, the UGT maybe used in combination with another 1-3 UGT enzyme or enzymes having apreference of specificity for certain substrates. For example, one UGTmay preferentially act as a 1-3 UGT on C13 glycosyl substrates whereasanother UGT enzyme preferentially acts as a 1-3 UGT on C19 glycosylsubstrates.

In some embodiments, a microbial cell expressing the 1-3 UGT enzyme isfed with RebD, and converts at least about 15%, or at least about 20%,or at least about 25%, or at least about 30%, or at least about 50%, orat least about 75%, or at least about 90% of the Reb D to Reb M. Invarious embodiments, such conversions are allowed to take place for atleast about 8 hours, or at least about 24 hours in some embodiments. Forexample, the conversion may be allowed to take place in the culture forfrom 8 hours to about 72 hours. In some embodiments, the conversion maybe allowed to take place for about 24 hours to about 60 hours. In someembodiments, the microbial cell converts at least about 40%, or at leastabout 50%, or at least about 75%, or at least about 90% of the Reb D toReb M in about 48 hours or less or about 24 hours or less.

In some embodiments, the microbial cell is fed with stevioside andexpresses the 1-3 UGT, and converts at least about 15%, or at leastabout 20%, or at least about 25%, or at least about 30%, or at leastabout 50% of the stevioside to Reb A. In some embodiments, the enzymeconverts at least about 75% or at least about 90% of the stevioside toRebA. In various embodiments, such conversions are allowed to take placefor at least about 8 hours, or at least about 24 hours in someembodiments. For example, the conversion may be allowed to take place inthe culture for from 8 hours to about 72 hours. In some embodiments, theconversion may be allowed to take place for about 24 hours to about 60hours. In some embodiments, the microbial cell converts at least about40%, or at least about 50%, or at least about 75%, of the stevioside toRebA in about 48 hours or less or about 24 hours or less.

While the native UGT enzymes are generally plant enzymes (which oftenhave optimal temperatures in the range of 20-24° C.) or are derived fromplant enzymes, the present disclosure in some embodiments enablesproduction of the glycosylated product at high yield in microbial cells(e.g., bacterial cells such as E. coli), with enzyme productivity attemperatures above 24° C., such as from 24° C. to 37° C., or from 27° C.to 37° C., or from 30° C. to 37° C. In some embodiments, culturing isconducted at from 30 to 34° C.

In some embodiments, the process is scalable for large-scale production.For example, in some embodiments, the size of the culture is at leastabout 100 L, at least about 200 L, at least about 500 L, at least about1,000 L, or at least about 10,000 L, or at least about 100,000 L, or atleast about 500,000 L.

In various embodiments, methods further include recovering glycosylatedproduct from the cell culture or from cell lysates. In some embodiments,the culture produces at least about 100 mg/L, or at least about 200mg/L, or at least about 500 mg/L, or at least about 1 g/L, or at leastabout 2 g/L, or at least about 5 g/L, or at least about 10 g/L, or atleast about 20 g/L, or at least about 30 g/L, or at least about 40 g/L,or at least about 50 g/L of the glycosylated product, which in someembodiments is extracted from the culture media.

In some embodiments, the substrate is a terpenoid, such as amonoterpenoid, sesquiterpenoid, or triterpenoid. In some embodiments,the terpenoid is a triterpenoid, which is optionally mogrol or amogroside. In some embodiments, the 1-3 UGT enzyme of the presentdisclosure has broad substrate activity towards glycosides, aliphaticand branched alcohols, substituted phenols, flavonoids and gallates. Seee.g. Dewitt, G. et al., Screening of recombinant glycosyltransferasesreveals the broad acceptor specificity of stevia UGT-76G1, J.Biotechnology 233 (2016) 49-55. Substrates of the UGT enzyme may includeterpenoid glycosides (isoprenoids), including diterpenoid glycosidessuch as steviol glycosides and triterpenoid glycosides such asmogrosides.

In some embodiments, the method comprises growing the host cell in thepresence of the substrate. The substrate may be fed to the culture, orin some embodiments, the substrate is synthesized by the host cell. Insome embodiments, the substrate is steviol, and the host cell expressesa plurality of UGT enzymes to produce target steviol glycosides, such asRebM.

In some embodiments, the glycosylated products (e.g., RebM) are purifiedfrom media components. Thus, in some embodiments, the methods compriseseparating growth media from E. coli cells, and isolating the desiredglycosylation products (e.g, RebM) from the growth media. In someembodiments, product such as RebM is further extracted from the cellularmaterial.

In some aspects, the invention provides methods for making a productcomprising a glycosylated product, such as RebM. The method comprisesincorporating the target steviol glycoside (produced according to thisdisclosure) into a product, such as a food, beverage, oral care product,sweetener, flavoring agent, or other product. Purified steviolglycosides, prepared in accordance with the present invention, may beused in a variety of products including, but not limited to, foods,beverages, texturants (e.g., starches, fibers, gums, fats and fatmimetics, and emulsifiers), pharmaceutical compositions, tobaccoproducts, nutraceutical compositions, oral hygiene compositions, andcosmetic compositions. Non-limiting examples of flavors for which RebMcan be used in combination include lime, lemon, orange, fruit, banana,grape, pear, pineapple, mango, bitter almond, cola, cinnamon, sugar,cotton candy and vanilla flavors. Non-limiting examples of other foodingredients include flavors, acidulants, and amino acids, coloringagents, bulking agents, modified starches, gums, texturizers,preservatives, antioxidants, emulsifiers, stabilizers, thickeners andgelling agents.

In some aspects, the invention provides methods for making a sweetenerproduct comprising a plurality of high-intensity sweeteners, saidplurality including two or more of a steviol glycoside, a mogroside,sucralose, aspartame, neotame, advantame, acesulfame potassium,saccharin, cyclamate, neohesperidin dihydrochalcone, gnetifolin E,and/or piceatannol 4′-O-β-D-glucopyranoside. The method may furthercomprise incorporating the sweetener product into a food, beverage, oralcare product, sweetener, flavoring agent, or other product, includingthose described above.

Target steviol glycoside(s), such as RebM, and sweetener compositionscomprising the same, can be used in combination with variousphysiologically active substances or functional ingredients. Functionalingredients generally are classified into categories such ascarotenoids, dietary fiber, fatty acids, saponins, antioxidants,nutraceuticals, flavonoids, isothiocyanates, phenols, plant sterols andstanols (phytosterols and phytostanols); polyols; prebiotics,probiotics; phytoestrogens; soy protein; sulfides/thiols; amino acids;proteins; vitamins; and minerals. Functional ingredients also may beclassified based on their health benefits, such as cardiovascular,cholesterol-reducing, and anti-inflammatory.

Further, target steviol glycoside(s), such as RebM, and sweetenercompositions obtained according to this invention, may be applied as ahigh intensity sweetener to produce zero calorie, reduced calorie ordiabetic beverages and food products with improved tastecharacteristics. It may also be used in drinks, foodstuffs,pharmaceuticals, and other products in which sugar cannot be used. Inaddition, RebM and sweetener compositions can be used as a sweetener notonly for drinks, foodstuffs, and other products dedicated for humanconsumption, but also in animal feed and fodder with improvedcharacteristics.

Examples of products in which target steviol glycoside(s) and sweetenercompositions may be used include, but are not limited to, alcoholicbeverages such as vodka, wine, beer, liquor, and sake, natural juices,carbonated soft drinks, diet drinks, zero calorie drinks, reducedcalorie drinks and foods, yogurt drinks, instant juices, instant coffee,powdered types of instant beverages, canned products, syrups, fermentedsoybean paste, soy sauce, vinegar, dressings, mayonnaise, ketchups,curry, soup, instant bouillon, powdered soy sauce, powdered vinegar,types of biscuits, rice biscuit, crackers, bread, chocolates, caramel,candy, chewing gum, jelly, pudding, preserved fruits and vegetables,fresh cream, jam, marmalade, flower paste, powdered milk, ice cream,sorbet, vegetables and fruits packed in bottles, canned and boiledbeans, meat and foods boiled in sweetened sauce, agricultural vegetablefood products, seafood, ham, sausage, fish ham, fish sausage, fishpaste, deep fried fish products, dried seafood products, frozen foodproducts, preserved seaweed, preserved meat, tobacco, medicinalproducts, and many others.

During the manufacturing of products such as foodstuffs, drinks,pharmaceuticals, cosmetics, table top products, and chewing gum, theconventional methods such as mixing, kneading, dissolution, pickling,permeation, percolation, sprinkling, atomizing, infusing and othermethods may be used.

Aspects and embodiments of the invention are now described withreference to the following examples.

EXAMPLES

Aspects and embodiments of the invention are now described withreference to the following examples.

Example 1: Screening of Mutants for MbUGT1-3_1 (SEQ ID NO: 5)

Plasmids comprising polynucleotides encoding UGT MbUGT1-3_1 (SEQ ID NO:5) and 95 selected mutants were transformed into E. coli cells (ΔushA,ΔgalETKM, Δpgi; overexpressed pgm, galU). The resulting strains wereseeded into a rich seed media and incubated overnight in 96-well platesat 250 rpm at 37° C. to make the seed cultures.

Production cultures were seeded with the seed cultures by adding 40 μLof the seed to 360 μL of fresh production media containing 0.5 mMStevioside and 0.5 mM Rebaudioside D. The production culture wassubsequently grown for 48 hours in 96-well plates at 250 rpm at 37° C.Products were quantified using a LC-MS QQQ.

The 95 mutants screened include the following (amino acid position ofthese mutants is with respect to MbUGT1-3_1 (SEQ ID NO: 5):

i) Substitution:

-   -   F11L, K13R, T16E, V18L, E19A, G48T, S62A, D73P, L89W, V93L,        Y94E, W99F, E100C, N106R, E115K, Y119T, Q122E, K130W, V133A,        V133S, M136K, S153L, G164R, R165N, R166G, I169L, F200L, F204H,        F204P, K206A, K208D, K208N, F219D, F219S, L221P, R229A, I230F,        L233I, G241A, R243M, P245S, I246L, R257D, E260A, L261K, L262Q,        L264S, D269E, E271P, S273A, R294P, L296I, S301N, F304S, H309F,        V310A, T327S, A333V, F336L, V341I, Y348T, Q352A, Q352D, Q352E,        A354S, A354T, K355Y, L358I, M361Q, I362V, S375T, E383F, V387L,        I388E, I388S, R389Q, L400F, L404F, A406S, D413E, V418C, P426A,        S428K, S428G, V441I, D445E, D455N, Q457G, Q457K/S458Q, or Q457K.

ii) Substitution and Deletion:

-   -   Q457K and delete 5458 (ΔS458).

iii) Deletion:

-   -   ΔG159; ΔG159ΔG161; or ΔG159ΔS160ΔG161.

iv) Insertion:

-   -   Insertion of S between position 158 and 159 or insertion of K        between 456 and 457.

All of the 95 mutants of MbUGT1-3_1 were screened for their ability toproduce RebA and RebI as well as RebM from Stevioside (substrate). Table5 shows the fold improvement of selected mutants of MbUGT1-3_1 toproduce RebA and RebI (through 1-3 glucosylation at C-13-glucose) aswell as RebM (through 1-3 glucosylation at C-19-glucose). In Table 5,column 2 generally corresponds to glycosylation at the C13-glucose ofthe substrate, while column 3 generally corresponds to glycosylation atthe C19-glucose of the substrate. As shown, C-19 glycosylation isenhanced more than C-13 glycosylation. The mutant constructed with themutations in Table 5 is referred to herein as MbUGT1-3_2 (SEQ ID NO: 6).

TABLE 5 MbUGT1-3_1 (lead mutations) (RebA + RebI)/ (RebA + RebI +RebM/(RebD + Mutation Stevioside) RebM + RebE) Deletion 1.13 1.25G159_S160_G161 (ΔG159ΔS160ΔG161) L262Q 1.12 1.19 R294P 1.22 1.21 D413E1.15 1.17

Example 2: Bioconversion by 1-3′ Glycosylating Enzymes

Plasmids comprising polynucleotides encoding three UGTs, SrUGT76G1-L200A(SEQ ID NO: 1), MbUGT1-3_0 (SEQ ID NO: 4), MbUGT1-3_1 (SEQ ID NO: 5),and MbUGT1-3_2 (SEQ ID NO: 6) were individually transformed into E. colicells (ΔushA, ΔgalETKM, Δpgi; overexpressed pgm, galU). The resultingstrains were seeded into culture media containing 1 mM stevioside or 1mM RebD. After growth, steviol glycosides were recovered from theculture media. Strains were grown overnight in 96-well plates at 250 rpmat 37° C. The cells were then transferred to a fresh production cultureto 10% of the total volume. Each of stevioside (0.5 mM) and RebaudiosideD (0.5 mM) were included in the production culture. The productionculture was then grown for 48 hours in 96-well plates at 250 rpm at 37°C. Products were quantified using a LC-MS QQQ.

FIG. 4 shows the percent conversion of stevioside to RebA and thepercent conversion of RebD to RebM. The percent conversion for bothreactions was highest for MbUGT1-3_1 and MbUGT1-3_2, which have theamino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively.FIG. 5 shows the fold improvement of MbUGT1-3_1 and MbUGT1-3_2 comparedto SrUGT76G1-L200A.

Example 3: Screening of Mutants for MbUGT1-3_2 (SEQ ID NO: 6)

Plasmids comprising polynucleotides encoding UGT MbUGT1-3_2 (SEQ ID NO:6) and 96 selected mutants of MbUGT1-3_2 were transformed into E. colicells (ΔushA, ΔgalETKM, Δpgi; ΔaraA, overexpressed pgm, galU, UGT1-2enzyme). The resulting strains were seeded into a rich seed media andincubated overnight in 96-well plates at 250 rpm at 37° C. to make theseed cultures.

Production cultures were then seeded with the seed cultures by adding 40μL of the seed to 360 μL of fresh production media containing 1 g/Lstevioside/RebA leaf extract. The production culture was then grown for48 hours in 96-well plates at 250 rpm at 37° C. Products were quantifiedusing a UPLC-DAD.

The mutants screened include the following (amino acid position is withrespect to MbUGT1-3_1 (SEQ ID NO: 5):

i) Substitution:

-   -   T16G, V18L, E19A, D23E, R31K, C64S, 170V, L76N, P79M, W99F,        D114E, R125L, V133A, S150D, T202M, F204E, Y212F, Y212H, R218L,        D227S, E228S, I230F, I230Y, L233I, G237S/P238A, P238S, L239E,        L239G, A240S, A240V, M242A, M242I, I246Y, I247F, A252I, D253E,        L261K, L264E, A265D, E271P, S273A, C274G, C274L, Y282W, N292K,        S302G, L303M, F306W, L312M, Q314L, L318W, P323L, E331A, K340R,        S346V, Y348F/S349D/N350T/W351L/Q352E/I353N,        Y348G/S349E/N350F/W351G/Q352E/I353K,        Y348T/S349N/N350E/W351P/Q352E/I353E, W351F/Q352E/A354G, W351P,        Q352D/A354G, Q352E, A354S, I362L, A367N, E383S, E385A, H403Y,        L404F, F419K, S428K, L431I, D445E, D445I, A450L, or Q457G.

ii) Substitution and Deletion:

-   -   D227T and Δ228 to 234

iii) Deletion:

-   -   ΔG158 to G161; ΔG159 to G161; ΔS160 to ΔG161; ΔG161; Δ228; Δ228        to 229; Δ228 to 230; Δ228-231; Δ228 to 232; Δ228 to 233; Δ228 to        234; Δ228 to 235; Δ228 to 236; Δ228 to 237; ΔY320 to P323; ΔD325        to K326; or ΔK326.

iv) Insertion:

-   -   Insert LEA between F25 and L26;

iv) Replace:

-   -   Swap Y348-I353 with INPQG

Table 6 shows the ability of selected mutants of MbUGT1-3_2 (SEQ ID NO:6) to produce RebM. Column 2 of Table 6 shows the fold improvement inthe percentage of RebM in the reaction product.

TABLE 6 MbUGT1-3_2 lead mutations Fold Improvement Mutation % RebMDelete E225-T232 11.1 (ΔE225 to T232) S72Q 1.25 A305C 3.51 Y345F 3.97L428I 2.48

The deletion of amino acids E225 to T232 produced a surprising 11 foldimprovement in the production of RebM. Based on homology modeling, aminoacids E225 to T232 appear to form a loop near the bound substrate.Deletion of amino acids E225-T232 may cause a shift in the substratespecificity of the UGT enzyme in favor of the RebD to RebM reaction. Thesequence having a deletion of E225-T232 loop and S72Q, A305C, Y345F andL428I mutations with respect to MbUGT1-3_2 (SEQ ID NO: 6) is referred toas MbUGT1-3_3 (SEQ ID NO: 9).

Several deletions in the E225 to T232 region were tested, and these alsoshowed enhancements in RebM production. Table 7 shows various beneficialdeletions of the E225-T232 region (numbered according to SEQ ID NO:6)

TABLE 7 MbUGT1-3_2 Deletions Fold Improvement Mutation % RebM DeleteE225-T232 11.1 Delete E225-L230 3.2 Delete E225-H233 3.2 DeleteE225-P231 3.1 Delete E225-N229 1.7 Delete E225-S228 1.6 Delete E225 1.6Delete E225-I227 1.5 Delete E225-G234 1.5

SEQUENCES SEQ ID NO: 1 UGT76G1 [Stevia rebaudiana] (SEQ ID NO: 1)MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLES LVSYISSL SEQ ID NO: 2MbUGT1-3 Reference: US 2017/0332673MANWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLK VKDIKSAYSSEQ ID NO: 3 UGT76G1 L200A Reference: US 2017/0332673MAENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIAKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLE SLVSYISSLSEQ ID NO: 4 MbUGT1-3_0MAKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIAKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFL EIARGLVDSSEQ ID NO: 5 MbUGT1-3_1MAFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHGGWNSTLESVCEGVPMIFSDEGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRLMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLGSGGSGGSGRRRRIILFPVPFQGHINPMLQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIAKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIA RGLVDSQS SEQ ID NO: 6MbUGT1-3_2 MAFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHGGWNSTLESVCEGVPMIFSDEGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRLMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLGSGGSGRRRRIILFPVPFQGHINPMLQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELQMLASEEDEEVSCLITDALWYFAQSVADSLNLPRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIAKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLEHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGL VDSQS SEQ ID NO: 7Linker GSGGSG SEQ ID NO: 8 Linker GSGGSGGSG SEQ ID NO: 9 MbUGT1-3_3MAFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHGGWNSTLESVCEGVPMIFQDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRLMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLGSGGSGRRRRIILFPVPFQGHINPMLQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDHGPLAGMRIPIINEHGADELRRELELQMLASEEDEEVSCLITDALWYFAQSVADSLNLPRLVLMTSSLFNFHCHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAFSNWQIAKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLEHDRIVFQWLDQQPPSSVIYVSFGSTSEVDEKDFLEIARGLVDSQS

The invention claimed is:
 1. A uridine diphosphate-dependentglycosyltransferase (UGT) enzyme comprising an amino acid sequence thatis at least 85% identical to the amino acid sequence of SEQ ID NO: 5 orSEQ ID NO: 6, wherein: the enzyme has an insertion with respect to SEQID NO: 1 of a flexible and hydrophilic sequence of 6 to 12 amino acidsthat is predominately Glycine and Serine residues after the positioncorresponding to position 155 of SEQ ID NO: 5; and the enzyme retainsone or more of: a glycine (G) at the position corresponding to position54 of SEQ ID NO: 5; a leucine (L) at the position corresponding toposition 111 of SEQ ID NO: 5; and a methionine (M) at the positioncorresponding to position 183 of SEQ ID NO:
 5. 2. The enzyme of claim 1,wherein the amino acid sequence is at least 95% identical to SEQ ID NO:5 or SEQ ID NO:
 6. 3. The enzyme of claim 1, wherein the enzyme has Glyat the position corresponding to position 54 of SEQ ID NO: 5, Leu at theposition corresponding to position 111 of the SEQ ID NO: 5, and a Met atthe position corresponding to position 183 of SEQ ID NO:
 5. 4. Theenzyme of claim 1, wherein the insertion is GSGGSG (SEQ ID NO: 7) orGSGGSGGSG (SEQ ID NO: 8).
 5. The enzyme of claim 1, wherein the enzymecomprises a deletion of at least three amino acids corresponding toamino acids E225 to T232 with respect to the amino acid sequence of SEQID NO:
 6. 6. The enzyme of claim 5, wherein the enzyme comprises adeletion of the amino acids corresponding to amino acids E225 to T232 ofSEQ ID NO:
 6. 7. The enzyme of claim 1, wherein the enzyme comprises anamino acid substitution at one or more positions corresponding toposition 72, position 305, position 345, and position 428 of SEQ ID NO:6.
 8. The enzyme of claim 7, wherein the enzyme comprises one or moreamino acid substitutions selected from: a glutamine (Q) at the positioncorresponding to position 72 of SEQ ID NO: 6; a cysteine (C) at theposition corresponding to position 305 of SEQ ID NO: 6; a phenylalanine(F) at the position corresponding to position 345 of SEQ ID NO: 6; andan isoleucine (I) at the position corresponding to position 428 of SEQID NO:
 6. 9. A uridine diphosphate-dependent glycosyltransferase (UGT)enzyme comprising an amino acid sequence that is at least 85% identicalto the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, wherein theUGT enzyme comprises: a glycine (G) at the position corresponding toposition 54 of SEQ ID NO: 5; a leucine (L) at the position correspondingto position 111 of SEQ ID NO: 5; and a methionine (M) at the positioncorresponding to position 183 of SEQ ID NO:
 5. 10. A uridinediphosphate-dependent glycosyltransferase (UGT) enzyme comprising anamino acid sequence that has at least 85% sequence identity to the aminoacid sequence of SEQ ID NO:1, and comprising a deletion of amino acidscorresponding to E74 to T81 of SEQ ID NO:
 1. 11. A uridinediphosphate-dependent glycosyltransferase (UGT) enzyme that is acircular permutant of SrUGT76G1 (SEQ ID NO: 1), and having from 1 to 20amino acid modifications independently selected from amino acidsubstitutions, deletions, and insertions with respect to thecorresponding position of SEQ ID NO:1, with the proviso that thecircular permutant has a deletion of amino acids corresponding to aminoacids E74 to T81 of SEQ ID NO:
 1. 12. The enzyme of claim 5, wherein theenzyme comprises a deletion of at least five amino acids correspondingto amino acids E74 to T81 with respect to the amino acid sequence of SEQID NO:
 6. 13. The enzyme of claim 7, wherein the enzyme comprises asubstitution of glutamine (Q) or asparagine (N) at the positioncorresponding to position 72 of SEQ ID NO:
 6. 14. The enzyme of claim 7,wherein the enzyme comprises a neutral hydrophilic amino acid selectedfrom cysteine (C), Serine (S), or Threonine (T) at the positioncorresponding to position 305 of SEQ ID NO:
 6. 15. The enzyme of claim7, wherein the enzyme comprises a substitution of phenylalanine (F) orTryptophan (W) at the position corresponding to position 345 of SEQ IDNO:
 6. 16. The enzyme of claim 7, wherein the enzyme comprises asubstitution of isoleucine (I), valine (V), or alanine (A) at theposition corresponding to position 428 of SEQ ID NO:
 6. 17. The enzymeof claim 7, wherein the enzyme comprises 2, 3, or 4 of the followingsubstitutions with respect to SEQ ID NO: 6: S72Q, A305C, Y345F, andL428I.
 18. A uridine diphosphate-dependent glycosyltransferase (UGT)enzyme comprising an amino acid sequence that is at least 85% identicalto the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, andcomprising a deletion of at least three amino acids corresponding toamino acids E225 to T232 with respect to SEQ ID NO:
 6. 19. The UGTenzyme of claim 18, wherein the enzyme comprises a deletion of at leastfive amino acids corresponding to amino acids E225 to T232 with respectto SEQ ID NO:
 6. 20. The UGT enzyme of claim 18, wherein the enzymecomprises a deletion of the amino acids corresponding to amino acidsE225 to T232 with respect to SEQ ID NO:
 6. 21. The UGT enzyme of claim18, wherein the enzyme is at least 90% identical to SEQ ID NO: 5 or SEQID NO:
 6. 22. The UGT enzyme of claim 18, wherein the enzyme is at least95% identical to SEQ ID NO: 5 or SEQ ID NO:
 6. 23. A polynucleotideencoding the enzyme of any one of claims 1, 9, 10, 11 or
 18. 24. Anisolated recombinant microorganism comprising the polynucleotide ofclaim
 23. 25. A method for transferring a monosaccharide group to asubstrate comprising contacting a NDP-sugar and the substrate with theisolated recombinant microorganism of claim
 24. 26. A method fortransferring a glycosyl group to a substrate comprising growing theisolated recombinant microorganism of claim 24 in the presence of thesubstrate.